The development of the heart and body wall muscles of the Drosophila embryo provides a powerful model for investigating transcriptional regulatory networks and organ formation, with wide applicability of the findings to other biological systems, including comparable processes that occur during human development. We are taking an interdisciplinary, approach to this problem by combining genetics, genomics, biochemical, molecular, cellular and computational methods that enable us to obtain a systems-level understanding of organogenesis.[unreadable] Both the heart and somatic muscles are mesodermal derivatives arising from progenitor cells that undergo progressive determination under the influence of a well-orchestrated set of intrinsic and extrinsic regulatory influences. The former include the tissue-restricted transcription factors (TFs), Twist (Twi) and Tinman (Tin), while the latter include intercellular signaling mediated by Wnt, BMP, receptor tyrosine kinase (RTK)/Ras and Notch pathways. Our prior studies support a model in which these generic signals yield mesoderm-specific outputs by functioning together with Twi and Tin to regulate the expression of target gene enhancers. To test and extend this hypothesis, we have turned to a more comprehensive analysis of the transcriptional regulatory networks that govern muscle and heart development.[unreadable] We first define large sets of genes that are co-expressed in subpopulations of mesodermal cells using microarray-based gene expression profiling followed by whole-mount embryo in situ hybridization to validate the microarray results at cellular resolution. Wild-type embryos and those with genotypes causing informative developmental perturbations are used in these studies. Next, we determine the molecular mechanisms by which specific genes are co-regulated using a combination of computational and experimental methods. The former approach involves searching the entire Drosophila genome for evolutionarily conserved co-occurrences of DNA binding sites for TFs that are known or potential mesodermal regulators. Where necessary, the binding specificity of a TF is determined using a protein binding microarray (PBM) technology that was developed by our collaborators. Homotypic and heterotypic binding site clusters identified in genome-wide computational scans represent candidate cis-regulatory modules. In addition, statistical overrepresentation of particular sequence motifs found in association with sets of co-expressed genes suggests that the corresponding factors comprise a transcriptional code controlling the expression of these genes. Predicted CRMs and regulatory codes are then validated using transgenic reporter assays. [unreadable] Applying this integrative strategy in an iterative manner, we have elucidated the transcriptional regulatory network of one type of embryonic myoblast, the muscle founder cell (FC). Our initial work established that transcription of a subset of FC genes is regulated by a core code that includes a Ras-activated Ets domain TF in combination with Twi and Tin. However, these three TFs alone cannot account for the unique gene expression patterns of individual FCs. The muscle-specific homeodomain (HD) proteinsincluding Slouch (Slou) and Muscle segment homeobox (Msh)comprise one class of candidate FC specificity factors. Recently, we have acquired strong evidence that HD TFs indeed contribute to the transcriptional codes of individual FCs, thereby providing a molecular explanation for how these factors function as determinants of cellular identity.[unreadable] We initially showed that a small set of previously characterized FC genes is differentially responsive to Slou and Msh. We then identified many more HD-regulated genes on a genome-wide scale by expression profiling purified mesodermal cells from embryos in which an individual HD TF is overexpressed. Unexpectedly, Slou and Msh also activated genes expressed in fusion-competent myoblasts (FCMs), which do not normally express these TFs. These results suggest that HD TFs regulate two distinct temporal waves of muscle gene expression, one in the developing FC, and a second in the mature multinucleated myotube following myoblast fusion. [unreadable] Next, we used computational algorithms to determine that the inclusion of PBM-derived Slou or Msh binding sites within the FC core code of Ets, Twi and Tin defines a more specific combination of co-regulatory TFs for the corresponding sets of HD-responsive genes. While most binding sites are shared by Slou and Msh (HD-common motifs), each of these TFs also binds a few unique sequences (Slou- or Msh-specific motifs). Computational scans for combinations of motifs that are overrepresented in the noncoding regions of known Slou-responsive FC genes showed a statistical enrichment of both HD-common and Slou-specific sites, again clustered with the core set of FC TFs. In addition, Slou-specific sites were enriched among a larger set of FC genes for which Slou-responsiveness has not been assessed. Collectively, these results suggest that additional FC genes are regulated by Slou, and that this TF may exert different regulatory effects through distinct binding preferences. Indeed, mutagenesis of HD-common sites completely inactivated known FC CRMs, whichsince each HD TF is only expressed in a subset of cells in which target genes are transcribedis also consistent with multiple cell-specific HD TFs functioning through these common sites. Additional experiments are in progress to assess whether HD-specific sites also contribute to the cell-restricted activities of FC enhancers. [unreadable] To further investigate the combinatorial complexity of FC gene regulation, we are examining the involvement of the HD TF, Six4, and its cofactor Eyes absent (Eya). By genome-wide expression profiling and in situ hybridization, we found that FC genes can be activated, repressed or remain unaffected by mesodermal overexpression of Eya. Six4 binding specificity was determined by PBM analysis, and an overrepresentation of Six4 motifs was found in association with Eya-responsive FC genes. Slou binding sites are also enriched among combinations of TFs that include Six4, consistent with a combinatorial model of FC gene regulation in which multiple HD TFs act in concert. Experiments are in progress to test whether FC gene enhancers are directly regulated by Eya-Six4. Other current work involves profiling TF binding site occupancy in chromatin from purified mesodermal cells.[unreadable] Taken together, the above studies provide new insights into the transcriptional codes regulating muscle gene expression and into the roles of individual HD TFs that specify cellular identity. Our expression profiling studies also identify large numbers of target genes that serve as downstream mediators of differentiation and morphogenesis. A complete understanding of developmental regulatory networks requires knowledge of how these effector genes function in specific biological processes. To address this question, we have undertaken an RNAi screen of muscle and heart gene functions in mesoderm development. Among numerous cardiac phenotypes identified in whole-embryo RNAi experiments, we are focusing on a group of genes that are required for proper closure of the heart tube. We now have over a dozen genes that are associated with an RNAi-induced open-heart phenotype, with additional candidates remaining to be analyzed. Current efforts are directed at refining the cellular basis of this loss-of-function phenotype, and to determining if open-heart genes act together in one or more morphogenetic pathways.[unreadable] In summary, our investigations provide new insights into the regulatory networks that orchestrate specific aspects of Drosophila embryogenesis, and serve as an instructive experimental paradigm for related investigations in other developmental systems.